Oxytocin Analysis from Human Serum by LCMS after Derivatization

Abstract

BACKGROUNDOxytocin (OT) needs to be measured accurately to research its associations with social behaviors, conditions, and diseases in humans. Available enzyme‐linked immunosorbent assays lack the sensitivity to measure the very low (pg/mL) circulating levels in non‐pregnant/‐lactating humans (NPLH) reliably while radioimmunoassays are accurate but involve hazardous radioactive materials. One‐dimensional liquid chromatography mass spectrometry (LCMS) methods are currently not sensitive enough. Our recently developed LCMS method (Franke, 2018 PMID 30091853) can quantitate serum OT down to 10 pg/mL but precludes quantification in NPLH, which is known to be < 5 pg/mL. Thus, we sought to improve the lower limits of detection (LLOD) of OT measurements by forming OT products with higher lipophilicity thereby increasing the MS sensitivity as well as achieving better liquid‐liquid extraction (LLE) or solid‐phase extraction (SPE) outcomes.METHODS200 uL aq. OT (0.01–10 ng/mL) was reduced using 1,4‐dithiothreitol(DTT) or tris(2‐carboxyethyl)phosphine (TCEP) to cleave the dithio ether followed by alkylation with N‐alkyl iodoacetamide using alkyl chains (C10 to C18) according to Williams et al. 2009 (PMID 19734056). A second strategy involved derivatization of the phenolic tyrosine hydroxyl with biphenyl sulfonyl or dansyl chloride. The resulting products were then concentrated using LLE or SPE.RESULTSReduction using DTT or TCEP resulted in >95% cleavage to OT‐dithiol. Subsequent alkylation products derived from N‐alkyl iodoacetamide alkyl chains C10 and C12 were chosen over those with C14 or C16 for further analysis due to better product sensitivity. However, these products produced low yields and lower LCMS sensitivity than the underivatized OT. Enriching these products by LLE or SPE were also unsuccessful. Derivatization using biphenyl sulfonyl or dansyl chloride yielded mono‐tagged adducts of the reduced and unreduced OT but had lower LCMS sensitivity than the underivatized OT. Enriching these adducts with LLE or SPE did not improve LLOD via concentration.DISCUSSIONAlkylation of the thiol groups of the reduced OT with N‐alkyl iodoacetamide or sulfonylation of native OT with an array of sulfonyl chlorides led to products less sensitive than underivatized OT. Attempts for enrichment of these products by SPE and LLE were unsuccessful.CONCLUSIONImproving LCMS LLODs of OT by derivatization approaches were unsuccessful as was enrichment of these products by LLE or SPE. Current attempts to enrich native OT from human serum for the improvements of LLOD during LCMS analyses are presented.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.